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Introduction to Sizing DNA on an Agarose Gel

Introduction to Sizing DNA on an Agarose Gel. HC 70AL Spring 2009 April 2, 2009 By Kristin Gill. How Do We Determine the Size of a PCR Product?. Fractionate a PCR product along with a DNA ladder by gel electrophoresis

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Introduction to Sizing DNA on an Agarose Gel

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  1. Introduction to Sizing DNA on an Agarose Gel HC 70AL Spring 2009 April 2, 2009 By Kristin Gill

  2. How Do We Determine the Size of a PCR Product? • Fractionate a PCR product along with a DNA ladder by gel electrophoresis • Compare the migration distance of the PCR fragment to that of the DNA ladder

  3. What Is a DNA Ladder? • A DNA solution composed of many DNA fragments with different known lengths • A ladder is used as a reference to estimate the size of an unknown DNA fragment

  4. 12,216 bp 2,072 bp 1,500 bp 4,072 bp 3,054 bp 600 bp 2,036 bp 1,636 bp 1,018 bp 506/517 bp 100 bp 396 bp What Ladders Do We Use in This Laboratory? 100-bb ladder (Invitrogen) 1-kb ladder (Invitrogen)

  5. How Can We Estimate the Size of the Unknown DNA Fragment? • Create a “standard curve” of DNA ladder fragments and their migration distance using either a logarithmic graph or an Excel program • Estimate the size of the unknown DNA fragment based on its migration distance relative to the standard curve

  6. Creating a Standard Curve • Measure how far each band traveled using a metric ruler (cm) Example:

  7. Creating a Standard Curve • Plot the data points using an Excel program Logarithmic Trend line

  8. 875 How Is the Size of the Unknown DNA Fragment Determined? • Measure the distance traveled by the unknown fragment • Interpolate the size based on the standard curve Example: 2 cm Fragment size: 875 bp

  9. Why Do You Need to Know the Size of the PCR Product? • Sequencing! • The amount of DNA used in the sequencing reaction is determined according to the size of the fragment (in bp) recommended by Applied Biosystems’ protocol (Taken from Experiment 1: Intro. to General Molecular…..Page 1.21)

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